Sizing Trinucleotide Repeat Sequences by Single‐Molecule Analysis of Fluorescence Brightness

Abstract

Analyzing DNA or RNA strands at the single-molecule level is an important research area which has delivered new insights for basic science. [1][2][3] Single-molecule DNA detection can also have a positive impact on biosensing and molecular diagnosis [4, 5] by offering reduced reagent consumption and the ability to examine minimal sample volumes without PCR amplification.[6] Several fluorescence-based methods have been developed to sequence-specifically detect individual nucleic acid strands and to identify mutations. These methods are capable of uncovering variations in the copy numbers of mRNA strands, [7][8][9][10] relatively large genetic alterations such as DNA deletions of at least 2 kb, [11][12][13][14] or small sequence changes up to five bases long. [15][16][17] Herein, we present a new approach to derive sequence information from DNA strands several hundred bases long. Within the method, base-specific fluorescence labeling is combined with brightness measurements to infer the number of trinucleotide repeats in highly repetitive DNA strands at the single-molecule level. The approach fills a methodological gap left by existing technologies and illustrates that measuring the fluorescence brightness of individual DNA molecules is a valid strategy to obtain sequence-specific information.Existing fluorescence-based approaches derive different types of genetic data from specific modes of fluorescence information. For example, copy numbers of cDNA or RNA [7][8][9][10] as well as single-point mutations [18] can be ascertained by the presence and/or localization of fluorescent signals from sequence-specific probes. Furthermore, large gene rearrangements, such as deletions, insertions or gene repetitions, are determined by measuring the spatial distance between two fluorescence probes bound to elongated DNA strands. [11][12][13][14] Sequencing methods, on the other hand, are used for the analysis of short base stretches by tracking the temporal order of fluorescence signals stemming from the release [15] or incorporation of several fluorescence-labeled nucleotides. [16,17] Fluorescence brightness measurements have also been used to size the length of DNA restriction fragments to determine point mutations at restriction cleavage sites. [19] But there are few other single-molecule approaches which apply fluorescence brightness measurements to infer more extensive sequence information from DNA strands.In order to address this shortage we developed an approach which determines the number of times a specific base occurs within a gene region using brightness information. The strategy is based on 1) the tagging of a specific base with fluorescence dyes followed by 2) the measurement of the accumulated brightness of the labeled strands via single-molecule fluorescence microscopy. To demonstrate its biological application potential, the approach is tested using huntingtin sequences with CAG trinucleotide repeats. Huntington's disease and at least eight other inherited neurodegenerative diseases share CAG triplet repeat exp...

Microarrays and single molecules: an exciting combination

“…21 The high-speed scanning of slides was also applied to monitor the expression of cancer cell subpopulations 24 and to characterize the variety of DNA molecules with large genomic and tandem repeat mutations. 25,26 As a limitation, wide-eld uorescence microscopy can only detect individual molecules when they are spaced beyond the diffraction limit at low surface densities.…”

Chemical Tags Facilitate the Sensing of Individual DNA Strands with Nanopores

“…For example, the highly repetitive DNA regions in trinucleotide-expansion-disease genes could be sized by labeling the same base in all of the repeats. [22] The extension of the technology towards sequencing by measuring the ionic-current modulation for each base [1] would be very difficult to achieve due to the small distance between neighboring bases. This does not, however, limit the potential of this method, because the concept of slowing down DNA through chemical tags is new and can be applied to various related nanopore approaches.…”

Chemical Tags Facilitate the Sensing of Individual DNA Strands with Nanopores

“…With further improvements in the tags, such as, a decrease in the size of the linker and the length of the tags, it will also be possible to reduce the nucleotide distance between the tags and thereby detect multiple bases in biologically relevant DNA strands. For example, the highly repetitive DNA regions in trinucleotide‐expansion‐disease genes could be sized by labeling the same base in all of the repeats 22. The extension of the technology towards sequencing by measuring the ionic‐current modulation for each base1 would be very difficult to achieve due to the small distance between neighboring bases.…”